STUDIES WITH TRANSGENIC MICE EXPRESSING A CONSTITUTIVELY ACTIVE MUTANT M3 RECEPTOR[unreadable] We tested the hypothesis that long-term, persistent activation of beta-cell M3 mAChRs might improve glucose tolerance and ameliorate the metabolic deficits associated with the consumption of a high-fat diet. To achieve the selective and persistent activation of beta-cell M3 receptors in vivo, we generated transgenic mice that overexpressed the Q490L mutant M3 receptor in their pancreatic beta-cells (beta-M3-Q490L Tg mice). The Q490L point mutation is known to enable the M3 receptor to efficiently activate Gq-type proteins even in the absence of ACh. For control purposes, we also generated transgenic mice that overexpressed comparable levels of wild-type (WT) M3 receptors in their pancreatic beta-cells (beta-M3-WT Tg mice). The transgenic mice and their corresponding WT littermates were analyzed in a series of in vitro and in vivo metabolic studies.[unreadable] Isolated perifused islets prepared from beta-M3-Q490L Tg mice displayed greatly enhanced insulin release even in the absence of ACh. In contrast, islets prepared from beta-M3-WT Tg mice did not show this effect but exhibited a leftward shift in their ACh concentration response (insulin release) curves. In vivo studies demonstrated that beta-M3-Q490L Tg mice displayed greatly improved glucose tolerance, associated with increased serum insulin levels. Moreover, beta-M3-Q490L Tg mice proved to be resistant to diet-induced glucose intolerance and hyperglycemia. Similar results were obtained with beta-M3-WT Tg mice. These results suggest that agonist-mediated chronic activation of beta-cell M3 muscarinic receptors may represent a useful approach to boost insulin output and improve glucose tolerance in the long-term treatment of glucose intolerance and type 2 diabetes.[unreadable] [unreadable] STUDIES WITH TRANSGENIC MICE EXPRESSING MUTANT M3 RECEPTORS ENDOWED WITH DIFFERENT G PROTEIN-COUPLING PROPERTIES[unreadable] GPCRs are known to regulate many aspects of beta-cell function. The nature of these effects is largely determined by the identity of the downstream heterotrimeric G proteins activated by a specific GPCR. To assess the role of different G protein signaling pathways in regulating beta-cell function in vivo, it would be highly desirable to establish an experimental system that allows the activation of distinct G proteins in pancreatic beta-cells only, preferably in a temporally controlled fashion. To address this issue, we took advantage of the recent availability of a modified version of the M3 mAChR (Armbruster et al., PNAS, 104, 5163-8, 2007). This receptor, which we refer to as R1Gq, is no longer able to bind its endogenous ligand, ACh, but can be efficiently activated by nanomolar concentrations of clozapine-N-oxide (CNO), a pharmacologically inert metabolite of clozapine. Following CNO-mediated activation, R1Gq selectively stimulates G proteins of the Gq family. Additional mutational modification of R1Gq led to the development of R2Gs, which, similar to R1Gq, can be activated by nanomolar concentrations of CNO (but not by ACh), but selectively stimulates Gs. We next generated transgenic mice that selectively expressed R1Gq and R2Gs in pancreatic beta-cells at comparable levels. R1Gq and R2Gs mice did not differ from their WT littermates in body weight, and fasting insulin and blood glucose levels. In the absence of CNO, the dynamic glucose and insulin responses to an i.p. glucose challenge were similar for R1Gq, R2Gs, and WT mice. As expected, CNO administration did not affect glucose homeostasis in WT mice. In striking contrast, CNO treatment of randomly fed R1Gq and R2Gs mice led to pronounced, dose-dependent hypoglycemic responses. Following i.p. glucose administration, R1Gq and R2Gs mice co-injected with CNO exhibited greatly improved glucose tolerance and a marked augmentation of first- and second-phase insulin release, as compared to transgenic animals not treated with CNO. These novel mouse models should further our understanding of the in vivo roles of specific G protein signaling pathways in regulating beta-cell function. Given the large number of GPCRs expressed by pancreatic beta-cells, these findings should be of considerable therapeutic relevance. [unreadable] [unreadable] IDENTIFICATION OF FACTORS THAT MODULATE M3 RECEPTOR FUNCTION IN AN INSULINOMA CELL LINE[unreadable] We demonstrated that treatment of the MIN6 mouse insulinoma cell line with muscarinic agonists causes a striking increase in intracellular calcium levels, accompanied by a pronounced increase in insulin release. Interestingly, treatment of MIN6 cells with M3 receptor siRNA greatly reduced both the muscarinic agonist-induced increase in insulin release and the elevation of intracellular calcium levels. This observation strongly suggests that both responses are predominantly mediated by the M3 receptor subtype, similar to what we found with native beta-cells. The MIN6 mouse insulinoma cell line therefore represents an excellent in vitro model system to dissect the molecular pathways leading to M3 receptor-mediated insulin release. By using siRNA technology, we started to explore the molecular nature of the receptor kinases and RGS proteins that modulate M3 receptor function in these cells. We found, for example, that MIN6 cells express several RGS proteins at relatively high levels. RGS proteins are known to act as inhibitors of signal transduction cascades initiated by GPCRs because of their ability to increase the intrinsic GTPase activity of heterotrimeric G proteins. This GTPase accelerating activity leads to G protein deactivation and promotes desensitization. We used siRNA technology to knock down the expression of specific RGS proteins and receptor kinases in MIN6 cells. This work led to the identification of several RGS proteins and receptor kinases that exert an inhibitory effect on muscarinic agonist-induced insulin release in MIN6 cells. These findings suggest that agents that can selectively inhibit the function of these proteins may become therapeutically useful in enhancing beta-cell M3 receptor function in type 2 diabetes.